The perception of bacterial gene regulation has changed dramatically since the discoveries of repressor and activator proteins. We now know that gene regulation also involves many different types of transcriptional and translational control mechanisms that sense the level of small-molecule cellular effectors without the involvement of regulatory proteins. In many cases, the effectors are the nucleoside triphosphate (NTP) substrates of transcription, and their concentrations are sensed directly by RNA polymerase during transcription initiation and elongation. The principal objective of this study is to elucidate new and further characterize known mechanisms of bacterial gene regulation involving NTP-sensitive reiterative transcription during initiation and/or transcription start-site switching. These reactions produce alternative transcripts with different 5'-end sequences and different potentials for translation. Reiterative transcription is the repetitive addition of a nucleotide due to slippage between a nascent transcript and its template. Start-site switching is the selection of alternate sites of transcription initiation within the same promoter. The proposed studies will focus on Escherichia coli operons that encode proteins involved in pyrimidine biosynthesis or salvage. Our first aim is to define the mechanism of pyrimidine-mediated regulation of pyrG expression. The pyrG gene encodes CTP synthetase, which catalyzes the final step of the pyrimidine nucleotide biosynthetic pathway. Preliminary studies indicate that the levels of the initiating nucleotide CTP control pyrG expression through a mechanism involving limited CTP-dependent reiterative transcription and transcription start-site switching. The second aim is to continue our characterization of promoter sequences required for reiterative transcription or start-site switching and that dictate the various fates of the resulting transcripts. The results obtained will allow us to predict and manipulate the distinct mechanisms of reiterative transcription and start-site switching. Our third aim is to characterize mutant RNA polymerases selected for defective reiterative transcription in vivo. The analysis will include RNA polymerases containing non-active site rpoC mutations that inhibit non- productive reiterative transcription during initiation (not necessarily directly). Additionally, this analysis will include RNA polymerase mutants containing active site mutations selected for their abilities to either promote or inhibit reiterative transcription during elongation. The final aim is to map within RNA polymerase the path followed by transcripts produced by reiterative transcription. We will examine transcripts that cannot be productively elongated as well as transcripts that can switch to the normal elongation mode. The results from the last two aims should provide new information regarding the mechanics of RNA polymerase. The knowledge gained from our research should allow better predictions of mechanisms of gene regulation from genomic sequences, manipulation of bacteria for the purposes of biotechnology, the design of new therapies for the treatment of bacterial diseases, and a better understanding of the inner workings of all cells. PUBLIC HEALTH RELEVANCE: Bacterial gene regulation involves many different types of transcriptional and translational control mechanisms, including those in which small-molecule cellular effectors are sensed without the involvement of regulatory proteins. In this study, we will elucidate and characterize regulatory mechanisms in which effectors are the nucleoside triphosphate substrates of transcription, and their concentrations are sensed directly by RNA polymerase. Defining these mechanisms in molecular detail enhances our understanding of the inner workings of bacteria and increases our ability to control bacterial pathogens and to create new tools for biotechnology.